OBJECTIVE: Peptidylarginine deiminase 2 (PAD2) and PAD4 are expressed in the synovium of rheumatoid arthritis (RA) patients and catalyze citrullination of arginine residues in proteins targeted by anti-citrullinated protein antibodies (ACPAs). Little is known about the relative importance of PAD2 and PAD4 in generating citrullinated self-antigens. Here we investigate the ability of PAD2 and PAD4 to generate citrullinated targets for ACPAs in four human proteins. METHODS: Synovial fluid (SF) and plasma were collected from 42 RA patients. Human fibrinogen, human alpha-enolase (ENO1), human histone H3, and human serum albumin (HSA) were citrullinated in vitro by PAD2 or PAD4. The total degree of citrullination was determined using the anti-modified citrulline approach. Antibody binding to native and citrullinated proteins was measured by ELISA. RESULTS: ACPAs within pooled SF from multiple RA patients reacted equally well with, and cross-reacted with, PAD2- and PAD4-citrullinated fibrinogen. ACPAs from most individual patient SF and plasma samples bound equally well to PAD2- and PAD4-citrullinated fibrinogen or ENO1. When histone H3 was used as target, PAD4 was generally superior in generating epitopes recognized by ACPAs. No binding to citrullinated HSA was observed. CONCLUSION: In most patients, PAD2 and PAD4 are equally efficient in generating citrullinated target sites for ACPAs in fibrinogen and ENO1. The binding of autoantibodies to histone H3 was generally higher after citrullination with PAD4 than with PAD2. Citrullinated HSA is not a target for ACPAs.

• MeSH
• citrulinace MeSH
• fibrinogen imunologie   MeSH
• fosfopyruváthydratasa imunologie   MeSH
• histony imunologie   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• peptidylarginindeiminasa typu 2 MeSH
• peptidylarginindeiminasa typu 4 MeSH
• peptidylarginindeiminasy metabolismus   MeSH
• protilátky proti citrulinovaným peptidům metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• revmatoidní artritida enzymologie   imunologie   MeSH
• sérový albumin imunologie   MeSH
• synoviální tekutina MeSH
• techniky in vitro MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fibrinogen MeSH
• fosfopyruváthydratasa MeSH
• histony MeSH
• PADI2 protein, human MeSH Prohlížeč
• PADI4 protein, human MeSH Prohlížeč
• peptidylarginindeiminasa typu 2 MeSH
• peptidylarginindeiminasa typu 4 MeSH
• peptidylarginindeiminasy MeSH
• protilátky proti citrulinovaným peptidům MeSH
• rekombinantní proteiny MeSH
• sérový albumin MeSH
• vápník MeSH

The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis. The heterogeneous responses of RA patients to various biologics, even among ACPA-positive patients, emphasize the need for further stratification of the patients. We used high-density protein array technology for fingerprinting of ACPA reactivity. Identification of the proteome recognized by ACPAs may be a step to stratify RA patients according to immune reactivity. Pooled plasma samples from 10 anti-CCP-negative and 15 anti-CCP-positive RA patients were assessed for ACPA content using a modified protein microarray containing 1631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PADs) 2 and PAD4. IgG antibodies from anti-CCP-positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Curiously, the binding of IgG antibodies in anti-CCP-negative plasma was also enhanced by PAD2- and PAD4-mediated citrullination of 29 and 26 proteins, respectively. For only four proteins, significantly more ACPA binding occurred after citrullination with PAD2 compared to citrullination with PAD4, while the opposite was true for one protein. We demonstrate that PAD2 and PAD4 are equally efficient in generating citrullinated autoantigens recognized by ACPAs. Patterns of proteins recognized by ACPAs may serve as a future diagnostic tool for further subtyping of RA patients.

• MeSH
• autoantigeny krev   imunologie   MeSH
• biologické markery krev   MeSH
• čipová analýza proteinů MeSH
• citrulin metabolismus   MeSH
• citrulinace MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• peptidylarginindeiminasa typu 2 metabolismus   MeSH
• peptidylarginindeiminasa typu 4 metabolismus   MeSH
• protilátky proti citrulinovaným peptidům imunologie   MeSH
• revmatoidní artritida imunologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tandemová hmotnostní spektrometrie MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• autoantigeny MeSH
• biologické markery MeSH
• citrulin MeSH
• peptidylarginindeiminasa typu 2 MeSH
• peptidylarginindeiminasa typu 4 MeSH
• protilátky proti citrulinovaným peptidům MeSH

Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline, is involved in the breakage of self-tolerance in anti-CCP-positive rheumatoid arthritis. This reaction is catalyzed by peptidyl arginine deiminases (PADs), of which PAD2 and PAD4 are thought to play key pathogenic roles. Small-molecule PAD inhibitors such as the pan-PAD inhibitor BB-Cl-amidine, the PAD2-specific inhibitor AFM-30a, and the PAD4-specific inhibitor GSK199 hold therapeutic potential and are useful tools in studies of citrullination. Using an ELISA based on the citrullination of fibrinogen, we found that AFM-30a inhibited the catalytic activity of PADs derived from live PMNs or lysed PBMCs and PMNs and of PADs in cell-free synovial fluid samples from RA patients, while GSK199 had minor effects. In combination, AFM-30a and GSK199 inhibited total intracellular citrullination and citrullination of histone H3 in PBMCs, as determined by Western blotting. They were essentially nontoxic to CD4+ T cells, CD8+ T cells, B cells, NK cells, and monocytes at concentrations ranging from 1 to 20 μM, while BB-Cl-amidine was cytotoxic at concentrations above 1 μM, as assessed by flow cytometric viability staining and by measurement of lactate dehydrogenase released from dying cells. In conclusion, AFM-30a is an efficient inhibitor of PAD2 derived from PBMCs, PMNs, or synovial fluid. AFM-30a and GSK199 can be used in combination for inhibition of PAD activity associated with PBMCs but without the cytotoxic effect of BB-Cl-amidine. This suggests that AFM-30a and GSK199 may have fewer off-target effects than BB-Cl-amidine and therefore hold greater therapeutic potential.

• Klíčová slova
• cell viability, citrullination, peptidyl arginine deiminase (PAD), rheumatoid arthritis, small-molecule PAD inhibitors,
• MeSH
• aktivace enzymů MeSH
• histony metabolismus   MeSH
• inhibiční koncentrace 50 MeSH
• inhibitory enzymů farmakologie   MeSH
• lidé MeSH
• náchylnost k nemoci MeSH
• peptidylarginindeiminasa typu 2 antagonisté a inhibitory   metabolismus   MeSH
• peptidylarginindeiminasa typu 4 antagonisté a inhibitory   metabolismus   MeSH
• revmatoidní artritida etiologie   metabolismus   patologie   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histony MeSH
• inhibitory enzymů MeSH
• PADI2 protein, human MeSH Prohlížeč
• PADI4 protein, human MeSH Prohlížeč
• peptidylarginindeiminasa typu 2 MeSH
• peptidylarginindeiminasa typu 4 MeSH

We investigated the role of natural killer (NK) cells and CD161, their primary C-type-lectin-like receptor in rheumatoid arthritis (RA). Samples were compared with healthy donors (HD), dermatomyositic (DM), polymyositic (PM), and osteoarthritic (OA) patients. RA, PM, and DM NK cell cytotoxicities significantly decreased relative to the HD and OA NK cells (p<0.0001). These results correlated with an increased expression of NK cell inhibitory receptor CD161, in active disease RA patients. We demonstrated that NK cells are able to respond to mutated citrullinated vimentin (MCV), an RA-specific autoantigen, leading to increases in both PAD4 enzyme and CD161 mRNA expression. MGAT5 glycosidase involvement was detected in GlcNAc metabolism within the synoviocytes of RA patients. Our findings reveal a functional relationship between CD161 expression and NK cell cytotoxicity as well as reactivity to glycans and MCV, thus providing new insight into the pathogenesis of RA and confirming the involvement of surface glycosylation.

• MeSH
• autoantigeny farmakologie   MeSH
• autoimunitní nemoci imunologie   MeSH
• autoprotilátky krev   imunologie   MeSH
• buňky NK cytologie   účinky léků   imunologie   metabolismus   MeSH
• cytotoxicita imunologická účinky léků   imunologie   MeSH
• dermatomyozitida imunologie   MeSH
• dospělí MeSH
• exprese genu účinky léků   genetika   MeSH
• glukokortikoidy terapeutické užití   MeSH
• glykokonjugáty farmakologie   MeSH
• hydrolasy genetika   MeSH
• imunosupresiva terapeutické užití   MeSH
• lektinové receptory NK-buněk - podrodina B agonisté   genetika   metabolismus   MeSH
• leukocyty mononukleární cytologie   účinky léků   imunologie   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• N-acetylglukosaminyltransferasy genetika   MeSH
• osteoartróza imunologie   MeSH
• peptidylarginindeiminasa typu 4 MeSH
• peptidylarginindeiminasy MeSH
• počet buněk MeSH
• polymyozitida imunologie   MeSH
• polysacharidy farmakologie   MeSH
• revmatoidní artritida diagnóza   farmakoterapie   imunologie   MeSH
• senioři MeSH
• synoviální tekutina cytologie   metabolismus   MeSH
• vimentin farmakologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase MeSH Prohlížeč
• autoantigeny MeSH
• autoprotilátky MeSH
• glukokortikoidy MeSH
• glykokonjugáty MeSH
• hydrolasy MeSH
• imunosupresiva MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• N-acetylglukosaminyltransferasy MeSH
• PADI4 protein, human MeSH Prohlížeč
• peptidylarginindeiminasa typu 4 MeSH
• peptidylarginindeiminasy MeSH
• polysacharidy MeSH
• vimentin MeSH

INTRODUCTION: The aim of the study was to analyse genetic architecture of RA by utilizing multiparametric statistical methods such as linear discriminant analysis (LDA) and redundancy analysis (RDA). METHODS: A total of 1393 volunteers, 499 patients with RA and 894 healthy controls were included in the study. The presence of shared epitope (SE) in HLA-DRB1 and 11 SNPs (PTPN22 C/T (rs2476601), STAT4 G/T (rs7574865), CTLA4 A/G (rs3087243), TRAF1/C5 A/G (rs3761847), IRF5 T/C (rs10488631), TNFAIP3 C/T (rs5029937), AFF3 A/T (rs11676922), PADI4 C/T (rs2240340), CD28 T/C (rs1980422), CSK G/A (rs34933034) and FCGR3A A/C (rs396991), rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPA) and clinical status was analysed using the LDA and RDA. RESULTS: HLA-DRB1, PTPN22, STAT4, IRF5 and PADI4 significantly discriminated between RA patients and healthy controls in LDA. The correlation between RA diagnosis and the explanatory variables in the model was 0.328 (Trace = 0.107; F = 13.715; P = 0.0002). The risk variants of IRF5 and CD28 genes were found to be common determinants for seropositivity in RDA, while positivity of RF alone was associated with the CTLA4 risk variant in heterozygous form. The correlation between serologic status and genetic determinants on the 1st ordinal axis was 0.468, and 0.145 on the 2nd one (Trace = 0.179; F = 6.135; P = 0.001). The risk alleles in AFF3 gene together with the presence of ACPA were associated with higher clinical severity of RA. CONCLUSIONS: The association among multiple risk variants related to T cell receptor signalling with seropositivity may play an important role in distinct clinical phenotypes of RA. Our study demonstrates that multiparametric analyses represent a powerful tool for investigation of mutual relationships of potential risk factors in complex diseases such as RA.

• MeSH
• alely MeSH
• antigeny CD28 genetika   MeSH
• autoprotilátky genetika   MeSH
• dospělí MeSH
• genetická predispozice k nemoci genetika   MeSH
• genotyp MeSH
• interferonové regulační faktory genetika   MeSH
• jednonukleotidový polymorfismus genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• revmatoidní artritida genetika   MeSH
• revmatoidní faktor genetika   MeSH
• rizikové faktory MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD28 MeSH
• autoprotilátky MeSH
• interferonové regulační faktory MeSH
• IRF5 protein, human MeSH Prohlížeč
• revmatoidní faktor MeSH